Abstract

Polysialic acid (PSA) is a carbohydrate composed of a linear homopolymer
of α-2-8-linked sialic acid residues and is mainly attached to the
neural cell adhesion molecule (NCAM). Because of the large negative
charge of PSA, presence of PSA attenuates the adhesive property of NCAM
and increases the cellular motility. PSA expression on NCAM is
developmentally regulated, and PSA plays important roles in formation
and remodeling of the neural system through regulation of the adhesive
property of NCAM. Expression of the polysialated form of NCAM has been
also demonstrated in some malignant tumors, such as Wilms’ tumor and
small cell lung cancer. Despite the possible importance as an
oncodevelopmental antigen, however, significance of PSA expression in
most malignant tumors has not been revealed. Therefore, PSA expression
in non-small cell lung cancer was assessed in the present study. PSA
was expressed only in 5 (20.8%) of 24 pathological stage I cases,
whereas it was expressed in most stage IV cases (76.8%, 11 of 14
cases). PSA expression was correlated with nodal metastasis and distant
metastasis, but not with local extent of the primary tumor. Next,
expression of polysialyltransferase genes (PST and
STX genes) which controlled formation of PSA, was
examined. The PST gene was constantly expressed in both
normal lung tissue and tumor tissue of all cases. In contrast, the
STX gene was not expressed in normal lung tissue of any
case, and STX gene expression in tumor tissue was
closely correlated with tumor progression. The STX gene
was expressed only in 1 (4.2%) of 24 stage I cases, whereas it was
expressed in most stage IV cases (85.7%, 12 of 14 cases). These
results suggested that the PSA and STX
genes could be new targets of cancer therapy as well as important
clinical markers.

INTRODUCTION

Primary lung cancer is the leading cause of cancer death in
industrialized countries. Although
NSCLC
3
accounts for approximately 80% of primary lung cancer, recent advances
in the therapy have been limited
(1)
. Therefore,
establishment of more effective therapeutic modalities for NSCLC is
needed, for which biological features of NSCLC should be revealed more
clearly and discovery of a new target for the therapy is essential.

Carbohydrates expressing on cell surface play important roles in
cell-cell and/or cell-matrix interactions. PSA is a carbohydrate
composed of a linear homopolymer of α-2-8-linked sialic acid residues
and is mainly attached to the NCAM
(2, 3)
. Because of the
large negative charge of PSA, presence of PSA attenuates the adhesive
property of NCAM and removal of PSA increases the binding between
NCAM-expressing cells
(4)
. It is well known that PSA
expression on NCAM is developmentally regulated; although the
polysialilated form of NCAM (PSA-NCAM) is abundant in a variety of
embryonic tissues, the majority of NCAM in adult tissues lacks PSA. In
the embryonic brain, PSA expressed on NCAM reduces the NCAM adhesion,
which increases cellular motility and allows cell migration and neurite
outgrowth
(2, 3, 5)
. In the adult brain, although PSA is
lost from NCAM in most areas, PSA-NCAM is expressed only in the
hippocampus and olfactory bulb where structural and synaptic
rearrangement continue even in the adult
(2, 3)
. Thus, PSA
plays important roles in formation and remodeling of the neural system
through regulation of adhesive property of NCAM.

Reexpression of PSA-NCAM has been demonstrated in some malignant
tumors, although PSA is lost from NCAM in most adult tissues. Roth
et al.(6, 7)
revealed that PSA was expressed
and attached to NCAM in Wilms’ tumor. Expression of PSA-NCAM was also
found in neuroblastoma
(8)
, natural killer cell
derived-lymphoma
(9)
, and pancreatic carcinoma with neural
invasion
(10)
, suggesting that PSA-NCAM was closely
related to the development of some kinds of malignant tumors. Moreover,
expression of PSA-NCAM was found in SCLC, which was characterized by
high metastatic potential and rapid cell proliferation
(10)
. Scheidegger et al.(11)
established two sublines, E2 without PSA expression and E3 with PSA
expression, from a human SCLC-derived cell line (NCI-H69).
Interestingly, the PSA-positive E3 clone demonstrated high metastatic
potential, and the PSA-negative E2 clone had very low metastatic
potential, although a comparable amount of NCAM was expressed in E3 and
E2 clones. The results suggested that PSA allowed PSA-positive cancer
cells to detach from primary tumor by attenuating the adhesive property
of NCAM, and that PSA was involved with the metastatic potential.

However, there have been few reports on expression of PSA in other
malignant tumors, including NSCLC. Although Pujol et al.(12)
examined NCAM expression in NSCLC, they did not
report on PSA expression. Thus, significance of PSA expression in most
malignant tumors has not been revealed despite the possible importance
as an oncodevelopmental antigen, probably because tools available for
studying on PSA had been limited to antibodies against PSA and endo-N,
which specifically recognized and cleavaged PSA
(2)
.
However, two polysialyltransferases that synthesize PSA have been
recently cloned, and thereafter studies on PSA have been markedly
improved. It has been reported that these two polysialyltransferases,
PST [for human
(13)
, PST-1 for hamster
(14)
,
and ST8SiaII for mouse
(15)]
and STX [for human
(16,
17,
18)
, STX-1 for rat
(19)
, and ST9SiaIV
for mouse
(20)]
, can independently synthesize PSA.
Expression of polysialyltransferase genes is developmentally regulated;
both expression of the PST gene and that of the
STX gene are strong in the embryonic brain and attenuate in
the adult brain like expression of PSA
(13, 16, 18, 21)
.
However, there are some differences between the expression patterns of
the PST gene and that of the STX gene. In the
adult brain, the PST gene is abundantly expressed in the
amygdala, subthalamic nucleus, cerebral cortex, and occipital pole,
whereas the STX gene is strongly expressed in the
hippocampus, medulla oblongata, and putamen. In other human adult
tissues, the PST gene is expressed in a variety of tissues
(such as the heart, placenta, spleen, thymus, small intestine, and
peripheral blood leukocyte), whereas the STX gene is
expressed in limited tissues, such as the heart and thymus
(18)
. These results suggest that expression of the
PST gene and that of the STX gene are
independently regulated and that each polysialyltransferase might have
different biological roles. However, biological roles of each
polysialyltransferase, especially in development and progression of
malignant tumors, have not been revealed. In the present study, to
clarify the biological and clinical significance of PSA in NSCLC,
expression of PSA and that of polysialyltransferase genes in NSCLC were
examined.

MATERIALS AND METHODS

Patients and Tissue Samples.

Primary lung tumor tissues and corresponding normal lung tissues were
obtained from 57 patients with NSCLC and 4 patients with SCLC, during
operation at Kyoto University Hospital, after informed consent was
taken. No contamination of cancer cells in normal lung tissues was
confirmed microscopically. p-stage was determined by the current
tumor-node-metastasis classification, as revised in 1997
(22)
. Histological type was determined using the
classification by the WHO
(23)
. Samples were immediately
snap-frozen in liquid nitrogen and stored at −80°C until use. For
histological examination, samples were fixed in 10% (v/v) formalin and
then embedded in paraffin. Serial 4-μm sections were prepared from
each sample and served for H&E staining and IHS.

IHS.

To detect expression of PSA and NCAM, TSA-Indirect Kit (NEN Life
Science Products, Boston, MA), a sensitive IHS system was used
(24)
. To detect PSA, anti-PSA mAb 12F8 (rat IgM, 500μ
g/ml; PharMingen, San Diego, CA), which recognized specifically PSA
(18)
, was used as a primary antibody. To detect a
nonpolysialylated form of NCAM, anti-NCAM (CD56) mAbs 123C3 (mouse
IgG1, 1000 μg/ml; Zymed, South San Francisco, CA) and ERIC-1 (mouse
IgG1, 200 μg/ml; Santa Cruz Biotechnology, Santa Cruz, CA) were used.
All of the procedures were performed following the manufacture’s
protocol. To confirm “true” PSA expression, sections with and
without pretreatment of endo-N were used for every IHS for PSA
(24)
. For pretreatment of endo-N, sections were incubated
with endo-N (50 μg/ml), a kind gift from Dr. Y. Kawase (NGK
Insulators, Ltd., Handa, Aichi, Japan), for 60 min at 37°C
(6, 26, 27)
.

RNA Isolation and RT-PCR.

Total RNA was purified using the RNeasy mini kit (QIAGEN GmbH, Hilden,
Germany), following the manufacturer’s protocol. To digest
contaminated DNA, extracted RNA was incubated with DNase (Nippon Gene,
Toyama, Japan). RT of total RNA was performed using the Ready-To-Go
You-Prime First-Strand Beads and random hexomer (Amersham Pharmacia
Biotech, Piscataway, NJ), following the manufacturer’s protocol. For
accurate evaluation of expression of the STX and
PST genes using RT-PCR assay, cDNA aliquots were diluted at
more than four cDNA concentrations. Measurements were taken in a linear
phase of the reaction where cDNA concentration is directly proportional
to the signal intensity
(28)
.

The sense and antisense primers used for PCR amplification of the
STX gene were 5′-TCAAGCACAACATCCAGCCAG-3′ and
5′-AGGGGTTCATGGTTACCAGGTC-3′, which amplified a 399-bp fragment. The
sense and antisense primers used for PCR amplification of the
PST gene were 5′-ATGTGGAAAGGAGATTGACAG-3′ and
5′-AGTGTATACATGAGAAGACCTGT-3′, which amplified a 436-bp fragment
(20)
. PCR primers for the GAPDH gene, used as a
internal control, were purchased from Clontech Laboratories Inc. (Palo
Alto, CA), and the sequences were 5′-ACCACAGTCCAGCCATCAC-3′ and
5′-TCCACCACCCTGTTGCTGTA-3′, which amplify a 452-bp fragment. PCR
amplification was carried out with an initial 9 min of preincubation at
95°C to activate the AmpliTaq Gold (Perkin-Elmer Corp., Foster City,
CA), followed by the following profile: denaturation at 94°C for
30 s, annealing at 60°C for 60 s, and extension at 72°C
for 60 s. The number of cycles for STX or
PST gene amplification was 35, and that for GAPDH
gene amplification was 25. PCR products were electrophoresed on 2%
agarose gels, and gels were stained with ethidium bromide. The
identities of PCR products were confirmed by DNA sequencing. In every
PCR run, cDNA templates obtained from a resected human thymus tissue,
in which both STX and PST genes were expressed,
were used as a positive control, and tubes containing all ingredients,
except for cDNA templates, were also used as a negative control. In
addition, in every PCR run, it was confirmed that no PCR product was
amplified in tubes containing RNA samples without RT reaction.

Statistical methods.

Counts were compared by the χ2 test. Continuous
data were compared using Student’s t test if the
distribution of samples was normal, or using Mann-Whitney U
test if the sample distribution was asymmetrical. Differences were
considered significant when P was less than 0.05. All
statistical manipulations were performed using the SPSS for Windows
software system (SPSS Inc., Chicago, IL).

RESULTS

PSA Expression in Primary Lung Cancer.

PSA expression in primary lung cancer was examined
immunohistochemically. In all SCLC cases, positively stained signals
were demonstrated and the signals disappeared with pretreatment of
endo-N, which confirmed the “true” PSA expression (Fig. 1 and B⇓
). PSA expression proved to be positive
in 28 (49.1%) of 57 cases with NSCLC (Table 1
⇓
and Fig. 1 and E⇓
). The correlation between PSA
expression and the patients’ characteristics was analyzed in NSCLC
cases (Table 2)
⇓
and PSA expression proved to be closely correlated with p-stage
(P < 0.002). PSA expression was positive
only in 5 (20.8%) of 24 p-stage I cases, whereas it was positive in 9
(69.2%) of 13 stage III cases and 11 (78.6%) of 14 stage IV cases.
The correlation between PSA expression and each factor of tumor
progression [i.e., extent of primary tumor (T-factor),
nodal involvement (N-factor), or distant metastasis (M-factor)] was
also analyzed (Fig. 2
⇓
). Whereas no correlation between PSA expression and T-factor was
demonstrated, PSA expression was significantly correlated with N-factor
and M-factor. PSA was expressed more frequently in node-positive
(N1–3) cases (69.6%, 16 of 23 cases) than in negative (N0) cases
(35.3%, 12 of 34 cases; P = 0.011). PSA was
expressed in most cases with distant metastasis (78.6%, 11 of 14
cases), whereas it was expressed in only 17 of 43 (39.5%) cases
without distant metastasis (P = 0.011).

Expression of PSA and NCAM in primary lung cancer. IHS for
PSA using 12F8 as a primary antibody in a SCLC case (A)
and in a NSCLC, poorly differentiated adenocarcinoma case
(D), respectively. In IHS for PSA after treatment of
endo-N, which specifically digests PSA, positive signals seen in
A and D disappeared (B and
E), which confirmed the true PSA expression. NCAM
expression was strongly positive in the SCLC case (C)
and was moderately positive in the NSCLC case (F).

The correlation between PSA expression and tumor progression was
analyzed in each histological type. For squamous cell carcinoma or
large cell carcinoma, there proved to be no statistically significant
correlation, probably because of small number of cases with each
histological type. For adenocarcinoma, there proved to be a significant
correlation between PSA expression and p-stage (P = 0.013) or M-factor (P = 0.015). That
is, PSA expression was positive in 5 (20.8%) of 19 p-stage I, none
(0.0%) of one stage II, 6 (60.0%) of 10 stage III, and 8 (72.7%) of
11 stage IV cases with adenocarcinoma.

Analysis of correlation between PSA expression and tumor
differentiation revealed that PSA was expressed more frequently in
moderately to poorly differentiated tumor than in well-differentiated
tumor (Fig. 3
⇓
). Although PSA was expressed more frequently in squamous cell carcinoma
cases than in adenocarcinoma cases, the difference was probably due to
the fact that most adenocarcinomas were classified into
well-differentiated tumor.

PSA expression in NSCLC according to the grade of tumor
differentiation. PSA expression was positive in only 5 of 25 (20.0%)
cases with well-differentiated tumor, whereas it was positive in 15 of
20 (75.0%) and 8 of 12 (66.7%) cases with moderately and poorly
differentiated tumors, respectively (P < 0.001). Thus, PSA was positive in 23 of 32 (71.9%) cases with
moderately to poorly differentiated tumor. Large cell carcinoma was
classified into poorly differentiated tumor.

NCAM expression in NSCLC.

To clarify whether the carrier molecule of PSA expressed on NSCLC cells
was NCAM, expression of NCAM to which PSA was usually attached was
examined. IHS using 123C3 mAb revealed that NCAM expression was
positive in 9 of 57 (15.8%) NSCLC cases (Tables 1
⇓
2
⇓
3
⇓
and Fig. 1F⇓
). Although IHS using ERIC-1, another mAb against
NCAM, demonstrated almost the same staining pattern, the reactivity was
rather weak as compared with that of 123C3. That is, NCAM expression
was detected with ERIC-1 only in six of nine cases that showed positive
NCAM expression with 123C3. In the present study, analysis of NCAM
expression was performed based on results of IHS using 123C3.

Expression of NCAM according to the patients’ characteristics in NSCLC

In all of the NCAM-positive cases, PSA expression was also positive,
which suggested that NCAM was a carrier molecule in such cases.
However, NCAM expression was negative in the other 48 cases. Among the
NCAM-negative cases, PSA was expressed in 19 (39.6%) cases, which
suggested that PSA was attached to some molecules other than NCAM in
such cases (Table 4)
⇓
. Western blotting analysis was performed to reveal characteristics of
the carrier molecules of PSA. In some cases showing positive NCAM
expression by IHS, Western blotting analysis revealed a broad band with
a high molecular weight around Mr
200,000 that was digested with endo-N treatment, which seemed to
represent PSA-NCAM (data not shown). In other patients, no apparent
signal was demonstrated with Western blotting analysis, and
characteristics of the carrier molecules of PSA could not be revealed.

Comparison of the patients’ characteristics according to PSA
expression and NCAM

The correlation between the NCAM expression in NSCLC and the patients’
characteristics was analyzed. In contrast to PSA expression, NCAM
expression was not correlated with tumor progression (Table 3)
⇓
. With
respect to tumor differentiation, NCAM was expressed in only a few
cases with well-differentiated tumor (Fig. 4
⇓
).

NCAM expression in NSCLC according to the grade of tumor
differentiation. NCAM expression was positive in only 1 of 25 (4.0%)
cases with well-differentiated tumor, whereas it was positive in 4 of
20 (20.0%) and 4 of 12 (33.3%) cases with moderately and poorly
differentiated tumors, respectively. Thus, NCAM was positive in 8 of 32
(25.0%) cases with moderately to poorly differentiated tumor, and the
rate of positive NCAM expression was significantly higher than that in
well-differentiated tumor cases (P = 0.031). Large cell carcinoma was classified into poorly differentiated
tumor.

The patients’ characteristics were compared after all of the patients
were divided into the following three groups based on PSA and NCAM
status: NCAM-positive and PSA-positive groups (n = 9); NCAM-negative and PSA-positive groups
(n = 19); and NCAM-negative and PSA-negative
groups (n = 29; Table 4
⇓
and Figs. 5
⇓
6
⇓
7
⇓
). Interestingly enough, the NCAM-negative and PSA-positive groups, not
the NCAM-positive and PSA-positive groups, showed the most advanced
tumor extent. Most cases in the NCAM-negative and PSA-positive groups
had p-stage III or IV disease. In contrast, more than half of the cases
(19 of 29) in the NCAM-negative and PSA-negative groups had p-stage I
disease (Fig. 7
⇓
). These results strongly suggested that PSA, not NCAM,
played important roles in tumor progression, especially in nodal and/or
distant metastases.

Distribution of p-stage of NSCLC cases in negative PSA
expression and negative NCAM expression [PSA(−)/NCAM(−)] cases, in
positive PSA expression and negative NCAM expression[
PSA(+)/NCAM(−)] cases, and in positive PSA expression and positive
NCAM expression [PSA(+)/NCAM(+)] cases. Most (17 of 19) cases in the
PSA(+)/NCAM(−) group had p-stage IIIB or IV disease.

Expression of Polysialyltransferase Genes in Primary Lung Cancer.

In our preliminary experiment using Northern blotting analysis, there
proved to be almost no expression of the PST or
STX genes in normal lung or tumor tissue. In addition, it
has been already reported that expression of both polysialyltransferase
genes was attenuated in most adult tissues, including the lung
(19)
. Thus, RT-PCR was used in the present study (Fig. 8
⇓
). Interestingly, the PST gene was expressed in both normal
lung and tumor tissues of all SCLC and NSCLC cases, whereas
STX was not expressed in normal lung tissue of any case.
Expression of the PST gene in the normal lung was also
confirmed by RT-PCR using normal lung tissues obtained from
noncancerous patients as templates. STX gene expression in
tumor tissues was positive in 25 of 57 (43.9%) NSCLC cases, whereas it
was positive in all SCLC cases (Table 5)
⇓
.

Expression of STX gene according to the patients’ characteristics in
NSCLC

The correlation between STX gene expression and
characteristics of patients in NSCLC was analyzed. Expression of the
STX gene proved to be closely correlated with tumor
progression, although not correlated with histological type. The
STX gene was expressed in 10 of 13 (76.9%) p-stage III
cases and in 12 of 14 (85.7%) p-stage IV cases, whereas it was
expressed only in 1 of 24 (4.2%) p-stage I cases (Table 5)
⇓
. Moreover,
STX gene expression was significantly correlated with any
factor of tumor progression (T-, N-, or M-factor; Fig. 9
⇓
). The STX gene was expressed in most T3–4 cases (84.6%, 11
of 13 cases), in most N1–3 cases (73.9%, 17 of 23 cases), or in most
M1 cases (85.7%, 12 of 14 cases).

Finally, the correlation between expression of the STX gene
and expression of PSA/NCAM in NSCLC was analyzed. In all nine cases
with positive NCAM expression, PSA expression was also positive,
although the STX gene was expressed only in two of nine
(22.2%) cases. Of 48 cases without NCAM expression, STX
gene expression was positive in 23 cases and negative in 25 cases. PSA
was expressed only in 5 of 25 (20.0%) cases with NCAM-negative and
STX-negative expression, whereas it was expressed in 14 of
23 (60.9%) cases with NCAM-negative and STX-positive
expression. Thus, in contrast to NCAM-positive cases where PSA was also
positive regardless of STX gene expression, NCAM-negative
cases showed positive expression of PSA mostly when the STX
gene was expressed.

DISCUSSION

There have been no reports on PSA and polysialyltransferase in
malignant tumor, except some kinds of malignant tumor such as SCLC
(6,
7,
8,
9,, 29)
. It is well known that PSA increases motility
of the neural cells through attenuating the cell-cell and/or
cell-matrix adhesion. Moreover, it has been already reported that PSA
increases motility of SCLC cells and allows the cancer cells to detach
from the primary tumor, which causes formation of metastatic foci
(11)
. The present study, for the first time, revealed that
expression of PSA and a polysialyltransferase (STX) gene
were involved with tumor progression of NSCLC.

It has been already revealed using Northern blotting analysis that both
expression of the PST gene and that of the STX
gene are abundant in the embryonic lung and disappear in the adult lung
(18)
. However, RT-PCR analysis in the present study
revealed that the PST gene was expressed in the adult
normal lung tissues, although the STX gene was not.
Because the amount of RT-PCR products for the PST gene
was smaller, by far, than that for the GAPDH gene, it
was suggested that expression of the PST gene could not be
detected by Northern blotting. In cancer tissues, the STX
gene was expressed only in advanced-stage cases, whereas the
PST gene was expressed in all cases, which strongly
suggested that the STX gene played critical roles in tumor
progression of NSCLC. There have been few reports on the differences
between PST and STX(2, 19, 30)
, and
distinct roles of each polysialyltransferase remain unknown. There has
been no report on the reason why PST and/or STX
are expressed in some adult tissues such as the heart, or no report on
the roles of each polysialyltransferase during development of cancer.
In the present study, it was proved that expression of PSA in NSCLC was
closely correlated with expression of the STX gene. However,
it remains unknown whether PSA can be synthesized by STX
alone or by STX in cooperation with PST that is constantly
expressed in the normal lung.

According to previous reports, α-subunit of sodium channels is the
only carrier molecule of PSA other than NCAM in mammalian cells
(31)
; roles of PSA expressed on α-subunit of sodium
channels remain unknown. The present study revealed that 19 of 57 NSCLC
cases showed positive PSA expression, although negative NCAM
expression, suggesting that PSA can be attached to molecules other than
NCAM. Recently, Martersteck et al.(32)
have
identified Mr 180,000–260,000
proteins in RBL rat basophilic leukemia cells and MCF7 human breast
cancer cells as possible carrier molecules of PSA, which suggested that
PSA might be more widespread than originally believed. In addition,
Nakayama and Fukuda
(33)
reported that PSA could be
attached to fetuin and revealed that NCAM was not required necessarily
in PSA expression. Therefore, PSA can be expressed on some adhesion
molecules other than NCAM, especially during malignant formation, and
PSA may promote formation of metastases by attenuating the adhesive
properties of the “unknown” molecules expressed on NSCLC cells.

In all NCAM-positive cases, PSA expression was also positive regardless
of status of STX expression, which suggested that NCAM was a
good substrate to which PSA could be attached easily. That is, PSA may
be synthesized and attached to NCAM by PST alone,
independent of STX. In NCAM-negative cases, PSA expression
was almost positive if STX was expressed and was almost
negative if STX was not expressed. These results suggested
that PSA could not be synthesized and attached easily to carrier
molecules other than NCAM when PST alone was expressed.
Without NCAM expression, PSA might be synthesized only when
STX was expressed with or without PST expression.
Although carrier molecules of PSA in NSCLC should be examined in
additional studies, the present study clearly demonstrated that PSA
itself played critical roles in tumor progression, especially formation
of metastatic foci. Thus, PSA and STX can be new targets of
cancer therapy as well as important clinical markers in NSCLC.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

↵1 Supported by Grant-in-aid 11671317 (to F. T.)
for Scientific Research (C) from the Ministry of Education of Japan.